Anti-ALK (phospho Y1604) antibody [EP661Y] (ab51030)

Hi, Thank you very much! I will try the new one and see what happens. If you could tell me what PTN you used and what concentration you used when you prepared that example in your data sheet, that will be highly appreciated. Best,

ANSWER:

Thank you for contacting us.

Cells are serum-starved overnight, and then treated with 10 ng/ml PTN (Sigma P5333) for 10 min.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

The following is a brief protocol I used: SH-SY-5Y cells were seeded at 5 million cells per well in 6-well plate and incubate at 37 degree overnight. Then replace culture medium to starving medium with only 0.1% serum, and incubate 24 hours in 37 degree incubator; At the end of 24 hrs starvation, hrPTN from R&D were directly add to starving medium at 100 ng/ml, and incubate at 37 degree for 30 minutes. Then briefly wash cells with cold PBS, and add 300 ul 1X Cell Lysis Buffer with protease inhibitor cocktail from Cell Signaling and get the cell lysate; Cell lysate were run on reducing gel and block with 5% dry milk for 1 hr, then blot with anti-ALk phosph antibody overnight at 4 degree; Then wash with TBST for 3 times each for 10 minutes, Then blot with anti-rabbit secondary antibody for 1 hr, And then wash with TBST for 2 hrs with change of wash buffer every 20 minutes. Then develop the film. I tried 1:2000 dilution, without any signal, then I tried 1:500 dilution, still no any signal. What should I try?

ANSWER:

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

I appreciate the time you have spent on these experiments, and would like to offer a replacement, refund or credit note in compensation (providing the product has been purchased in the last 120 days). In order to arrange this, I would appreciate if you could confirm your order number and date of purchase?

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Hi, I bought anti-ALK (phosphor Y1604) antibody from you and tried it,but it does not work as you described. From your datasheet, you showed SH-SY-5Y cells treated with PTN, and detected ALK pY1604 at 220kDa, I tried it but I could not detect anything at that size, and I even diluted the antibody at 1:500. Could you do me a favor and tell me more details about your example experiment? Where did you get the PTN and what concentration you use?

ANSWER:

Thank you for contacting Abcam.

I am sorry to hear of the difficulties that you have been experiencing using these products. Would you mind sending me the details of your protocol. Images and any steps that you may have changed while troubleshooting are extrememly helpful as well.

These details will not only help me to better understand your difficulties and be able to offer suggestions but will also be used for internal testing of the products if deemed necessary. It will also needed if we should need to communicate with the lab regarding this issue. If we are unable to solve the problems you are experiencing we will replace or refund the antibody according to our Abpromise guarantee.

Please let me know if you have any questions.